A novel Atg5-shRNA mouse model enables temporal control of Autophagy in vivo

Macroautophagy/autophagy is an evolutionarily conserved catabolic pathway whose modulation has been linked to diverse disease states, including age-associated disorders. Conventional and conditional whole-body knockout mouse models of key autophagy genes display perinatal death and lethal neurotoxicity, respectively, limiting their applications for in vivo studies. Here, we have developed an inducible shRNA mouse model targeting Atg5, allowing us to dynamically inhibit autophagy in vivo, termed ATG5i mice. The lack of brain-associated shRNA expression in this model circumvents the lethal phenotypes associated with complete autophagy knockouts. We show that ATG5i mice recapitulate many of the previously described phenotypes of tissue-specific knockouts. While restoration of autophagy in the liver rescues hepatomegaly and other pathologies associated with autophagy deficiency, this coincides with the development of hepatic fibrosis. These results highlight the need to consider the potential side effects of systemic anti-autophagy therapies.     

Diet of bluegill Lepomis macrochirus in a South African reservoir during winter and summer

Alien fishes are considered a major threat to aquatic biodiversity in South Africa, yet relatively little regional information on their biology and ecology is available for many of these species. Seasonal changes in the diet of the bluegill Lepomis macrochirus in Howieson’s Poort Dam, Grahamstown, were assessed during summer and winter in 2014–2015, using stomach content analysis. In winter, juvenile and adult fish diets were dominated by crustacean zooplankton and insects, respectively. In summer, juvenile fish fed on crustaceans and insects, whereas adults consumed mostly fish eggs, indicating a potential impact by these invasive fish on native fish through oophagy.     

Dopamine but not l-dopa stimulates neural glutathione metabolism. Potential implications for Parkinson’s and other dopamine deficiency states

Dopamine is produced first by hydroxylalation of l-tyrosine to l-dihydroxyphenylalanine (l-dopa) and subsequently by the decarboxylation of l-dopa to dopamine catalysed by the enzymes tyrosine hydroxylase and aromatic l-amino acid decarboxylase (AADC) respectively. Reduced glutathione (GSH) acts as a major cellular antioxidant. We have investigated the role of dopamine in the control of GSH homeostasis in brain cells. The SH-SY5Y human neuroblastoma cell line was found to increase intracellular GSH levels in response to 50 μM dopamine treatment. Similarly the 1321N1 human astrocytoma cell line was found to increase GSH release in response to 50 μM dopamine. The same concentration of l-dopa was also found to increase intracellular GSH in SH-SY5Y cells, however when AADC was inhibited this affect was abolished. Furthermore 1321N1 cells which were found to have almost undetectable levels of AADC activity did not increase GSH release in response to 50 μM l-dopa. These results suggest that at these concentrations dopamine has the potential to act as a signal for the upregulation of GSH synthesis within neuronal-like cells and for the increased trafficking of GSH from astrocytes to neurons. This effect could potentially relate to the activation of antioxidant response elements leading to the induction of phase II detoxifying enzymes including those involved in GSH synthesis and release. The inability of l-dopa to produce a similar effect when AADC was inhibited or when AADC activity was absent indicates that these effects are relatively specific to dopamine. Additionally dopamine but not l-dopa treatment led in an increase in complex I activity of the respiratory chain in SH-SY5Y cells which may be related to the effect of dopamine on GSH levels.     

An improved method using densitometry for evaluating severity of laser photocoagulation induced CNV

Laser photocoagulation induced choroidal neovascularization currently is the most effective model available for the study of this disease in terms of efficacy of new drugs and therapies. Previously, evaluating the extent of choroidal neovascularization using this model was time- consuming and required the use of experienced personnel. We describe a new method for simple and rapid evaluation of laser induced choroidal neovascularization using densitometry. Fluorescein angiograms of a laser photocoagulated rat eye were scanned into a computer. Densitometry software subsequently was used to calculate the severity of the laser lesions. The densitometry method proved effective for calculating the extent of laser induced choroidal neovascularization. In addition, this method was more rapid than visual evaluations and less likely to produce errors.     

Crystal structures of human ADAMTS-1 reveal a conserved catalytic domain and a disintegrin-like domain with a fold homologous to cysteine-rich domains

The ADAMTS (a disintegrin-like and metalloproteinase domain with thrombospondin type I motifs) family of proteases plays a role in pathological conditions including arthritis, cancer, thrombotic thrombocytopenic purpura and the Ehlers-Danlos type VIIC and Weill-Marchesani genetic syndromes. Here, we report the first crystal structures for a member of the ADAMTS family, ADAMTS-1. Originally cloned as an inflammation-associated gene, ADAMTS-1 has been shown to be involved in tissue remodelling, wound healing and angiogenesis. The crystal structures contain catalytic and disintegrin-like domains, both in the inhibitor-free form and in complex with the inhibitor marimastat. The overall fold of the catalytic domain is similar to related zinc metalloproteinases such as matrix metalloproteinases and ADAMs (a disintegrin and metalloproteinases). The active site contains the expected organisation of residues to coordinate zinc but has a much larger S1' selectivity pocket than ADAM33. The structure also unexpectedly reveals a double calcium-binding site. Also surprisingly, the previously named disintegrin-like domain showed no structural homology to the disintegrin domains of other metalloproteinases such as ADAM10 but is instead very similar in structure to the cysteine-rich domains of other metalloproteinases. Thus, this study suggests that the D (for disintegrin-like) in the nomenclature of ADAMTS enzymes is likely to be a misnomer. The ADAMTS-1 cysteine-rich domain stacks against the active site, suggesting a possible regulatory role.     

The Effect of Organophosphate Exposure on Neuronal Cell Coenzyme Q10 Status

Neurochemical Research - Organophosphate (OP) compounds are widely used as pesticides and herbicides and exposure to these compounds has been associated with both chronic and acute forms of...     

Distribution of MT1 Melatonin Receptor Promoter-Driven RFP Expression in the Brains of BAC C3H/HeN Transgenic Mice

The pineal hormone melatonin activates two G-protein coupled receptors (MT₁ and MT₂) to regulate in part biological functions. The MT₁ and MT₂ melatonin receptors are heterogeneously distributed in the mammalian brain including humans. In the mouse, only a few reports have assessed the expression of the MT₁ melatonin receptor expression using 2-iodomelatonin binding, in situ hybridization and/or polymerase chain reaction (PCR). Here, we described a transgenic mouse in which red fluorescence protein (RFP) is expressed under the control of the endogenous MT₁ promoter, by inserting RFP cDNA at the start codon of MTNR1a gene within a bacterial artificial chromosome (BAC) and expressing this construct as a transgene. The expression of RFP in the brain of this mouse was examined either directly under a fluorescent microscope or immunohistochemically using an antibody against RFP (RFP-MT₁). RFP-MT₁ expression was observed in many brain regions including the subcommissural organ, parts of the ependyma lining the lateral and third ventricles, the aqueduct, the hippocampus, the cerebellum, the pars tuberalis, the habenula and the habenula commissure. This RFP-MT₁ transgenic model provides a unique tool for studying the distribution of the MT₁ receptor in the brain of mice, its cell-specific expression and its function in vivo.